Microorganism mutant conversion of sterols to androsta-4-ene-3,17-dione

ABSTRACT

Mutant which is used in a novel microbiological process to selectively degrade steroids having 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, to yield predominantly androst-4-ene-3,17-dione (AD) and small amounts of androsta-1,4-diene-3,17-dione (ADD). AD is a valuable intermediate to make useful steroids.

CROSS REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 849,525, filed Nov. 9, 1977now U.S. Pat. No. 4,293,644, which is a continuation-in-part of ourapplication Ser. No. 735,075 filed Oct. 22, 1976 and now abandoned.

BACKGROUND OF THE INVENTION

The transformation of steroids by microorganisms has been widely studiedand documented. Apparently, the earliest such work was by Mamoli andVercellone in 1937, Ber. 70, 470 and Ber. 70, 2079. They disclosed thereduction of 17-ketosteroids to 17β-hydroxysteroids by ferenting yeast.Since then, Peterson and Murray disclosed the 11α-hydroxylation ofprogesterone with the fungus Rhizopus nigricans; see, U.S. Pat. No.2,602,769 (1952). More recently, Kraychy et al. in U.S. Pat. No.3,684,657 (1972) discloses the selective microbiological degradation ofsteroidal 17-alkyls by fermenting a steroid containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 toprepare androst-4-ene-3,17-dione, androst-1,4-diene-3,17-dione, and20α-hydroxymethyl-pregna-1,4-dien-3-one. Even more recently, Marsheck etal. in U.S. Pat. No. 3,759,791 (1973) disclose the selectivemicrobiological preparation of androst-4-ene-3,17-dione by fermenting asteroid of the cholestane or stigmastane series containing at least 8carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805,which has been characterized as Mycobacterium vaccae.

BRIEF SUMMARY OF THE INVENTION

A mutant which is characterized by its ability to selectively degradesteroids having 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, and accumulate androst-4-ene-3,17-dione, hereinafter referredto as AD, in the fermentation beer. This mutant can be obtained frommicroorganisms of the following genera by using the mutation proceduresdisclosed herein or other mutation procedures: Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium,Nocardia, Protaminobacter, Serratia, and Streptomyces. A preferred genusis Mycobacterium. Exemplary species of this genus are M. phlei, M.smegmatis, M. rhodochrous, M. mucosum, M. fortuitum, and M. butyricum.Specifically exemplified kherein is the novel mutant microorganism,Mycobacterium fortuitum, NRRL B-11045 which is used to selectivelydegrade steroids having 17-alkyl chains of from 2 to 10 carbon atoms, toAD. Examples of suitable steroid substrates are sitosterols,cholesterol, stigmasterol, campesterol, and like steroids with 17-alkylside chains of from 2 to 10 carbon atoms, inclusive. These steroidsubstrates can be in either the pure or crude form.

DETAILED DESCRIPTION OF THE INVENTION

The Microorganism

A mutant which is characterized by its ability to selectively degradesteroids having 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, and accumulate AD in the fermentation beer can be obtained bymutating microorganisms of the following genera: Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium,Nocardia, Protaminobacter, Serratia, and Streptomyces. Mycobacteriumfortuitum, ATCC 6842, has been mutated, as disclosed herein, to give anovel laboratory mutant microorganism.

The 1974 ATCC Catalogue discloses the following alongside the listing ofATCC 6842: "J. C. Cruz 2. Cold abscess. Acta Med. Rio de Janeiro 1:1(1936). Medium 90 37C". M. fortuitum, ATCC 6842, degrades sterolsnon-selectively to small molecular weight compounds, e.g. CO₂ +H₂ O.Thus, this microorganism is not suitable as a selective steroiddegrader.

Mutation of M. fortuitum, ATCC 6842, using nitrosoguanidine has resultedin the production of a novel mutant which selectively degrades steroidshaving 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, toproduce AD. This mutant microorganism has been given the accessionnumber NRRL B-11045 by the Northern Regional Research Laboratory, U.S.Department of Agriculture, Peoria, Ill., U.S.A., where it has beendeposited in the permanent collection. A subculture of thismicroorganism is freely available from this depository by request madethereto. It should be understood that the availability of the culturedoes not constitute a license to practice the subject invention inderogation of patent rights granted with the subject instrument bygovernmental action.

The microorganism of the subject invention has been distinguished fromthe Mycobacterium species NRRL B-3805, disclosed in U.S. Pat. No.3,749,791, which is discussed supra. NRRL B-3805 has the generalcharacteristics of Mycobacterium vaccae which is a distinctly differentspecies than the M. fortuitum of the subject invention. See Bergey'sManual of Determinative Bacteriology, 8th Edition. The Williams andWilkins Company, 1974, on pages 695 and 696 for a comparison of thesemicroorganisms.

The morphology and drug sensitivities of M. fortuitum NRRL B-11045 isindistinguishable from that of the parent M. fortuitum, ATCC 6842. M.fortuitum is an acid-fast non-motile, non-spore-forming bacillibelonging to the family Mycobacteriaceae of the order Actinomycetales.According to Runyons classification, Runyon, E. H. 1959 Med. Clin. NorthAmerica 43: 273, M. fortuitum is a nonchromogenic group IVmycobacterium, i.e., it grows rapidly at low temperatures to producenonpigmented colonies on relatively simple media.

M. fortuitum ATCC 6842 and M. fortuitum NRRL B-11045 are clearlydistinguishable in their action on steroid molecules. As disclosedabove, M. fortuitum ATCC 6842 is a non-selective degrader of steroids,whereas M. fortuitum NRRL B-11045 is a selective degrader. This propertyof M. fortuitum NRRL B-11045 makes it highly useful, as disclosedherein.

The mutation of M. fortuitum ATCC 6842 to give a mutant which is thenremutated to give M. fortuitum NRRL B-11045 was accomplished by the useof nitrosoguanidine. The details of the procedure are described infra.Though the mutation and transformation procedures, disclosed herein, aredetaled for a Mycobacterium, it should be understood that similar orequivalent procedures can be used with microorganisms of the othergenera, as disclosed herein.

THE TRANSFORMATION PROCESS

The selective transformation of the subject invention can be effected ina growing culture of M. fortuitum NRRL B-11045 by either adding theselected steroid substrate to the culture during the incubation period,or incorporating it in the nutrient medium prior to inoculation. Thesteroid can be added singly or in combination with another steroid. Thepreferred, but not limiting, range of concentration of the steroid inthe culture is about 0.1 to about 100 grams per liter. The culture isgrown in a nutrient medium containing a carbon source, for example, anassimilable carbohydrate, and a nitrogen source, for example, anassimilable nitrogen compound or proteinaceous material. Preferredcarbon sources include glucose, brown sugar, sucrose, glycerol, starch,cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogensources include cornsteep liquor, yeast, autolyzed brewer's yeast withmilk solids, soybean meal, cottonseed meal, cornmeal, milk solids,pancreatic digest of casein, fish meal, distillers' solids, animalpeptone liquors, meat and bone scraps, ammonium salts and the like.Combinations of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like, need not be added to the fermentation mediasince tap water and unpurified ingredients are used as components of themedium prior to sterilization of the medium.

The transformation process can range from about 72 hours to 15 days ormore. The incubation temperature can range from about 25° C. to about37° C., with 30° C. being preferred for NRRL B-11045. The contents areaerated with sterilized air and agitated to facilitate growth of themicroorganism, and, thus, enhance the effectiveness of thetransformation process.

Upon completion of the transformation process, as evidenced by thinlayer chromatography using silica gel plates (E. Merck, Darmstadt) and asolvent system consisting of 2:3 (by volume) ethyl acetate-cyclohexane,the desired transformed steroid is recovered by means well known in theart. For example, the fermentation (transformation) reaction mixture,including the fermentation liquor and cells, can be extracted with awater-immiscible organic solvent for steroids. Suitable solvents aredichloromethane (preferred), methylene chloride, chloroform, carbontetrachloride, ethylene chloride, trichloroethylene, ether, amylacetate, benzene and the like.

Alternatively, the fermentation liquor and cells can be first separatedby conventional methods, e.g., filtration or centrifugation, and thenseparately extracted with suitable solvents. The cells can be extractedwith either water-miscible or water-immiscible solvents. Thefermentation liquor, freed of cells, can be extracted withwater-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtratevacuum distilled to dryness. The resulting residue containing thedesired transformed steroid then can be dissolved in a minimum of ethylacetate-cyclohexane (20:80). This solution then can be chromatographedon silica gel. AD can be separated from the silica gel by elution withthe solvent system ethyl acetate-chloroform (15:85). The compound thencan be isolated as a separate entity by evaporation of the solvent andrecrystallization from hexane.

The desired product of the subject invention transformation process isthe known steroid intermediate AD. This compound is useful as anintermediate in the synthesis of useful steroidal hormones. For example,AD can be used to make testosterone according to processes disclosed inU.S. Pat. Nos. 2,143,453; 2,253,798; 2,264,888 and 2,356,154.

The following examples are illustrative of the process and product ofthe subject invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Preparation of Mutant M. fortuitum NRRL B-11045 From M.fortuitum ATCC 6842.

(a) Nitrosoquinidine Mutagenesis

Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the followingsterile seed medium:

    ______________________________________                                        Nutrient Broth (Difco) 8 g/liter                                              Yeast Broth            1 g/liter                                              Glycerol               5 g/liter                                              Distilled Water, q.s.  1 liter                                                ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to sterilization at 121°C. for 20 minutes.

The cells are grown to a density of about 5×10⁸ per ml, pelleted bycentrifugation, and then washed with an equal volume of sterile 0.1 Msodium citrate, pH 5.6. Washed cells are resuspended in the same volumeof citrate buffer, a sample removed for titering (cell count), andnitrosoguanidine added to a final concentration of 50 μg/ml. The cellsuspension is incubated at 37° C. in a water bath for 30 minutes, afterwhich a sample is again removed for titering and the remaindercentrifuged down and washed with an equal volume of sterile 0.1 Mpotassium phosphate, pH 7.0. Finally, the cells are resuspended in asterile minimal salts medium, minus a carbon source, consisting of thefollowing:

    ______________________________________                                        NH.sub.4 NO.sub.3    1.0    g/liter                                           K.sub.2 HPO.sub.4    0.25   g/liter                                           MgSO.sub.4.7H.sub.2 O                                                                              0.25   g/liter                                           NaCl                 0.005  g/liter                                           FeSO.sub.4.7H.sub.2 O                                                                              0.001  g/liter                                           Distilled Water, q.s.                                                                              1      liter                                             ______________________________________                                    

The pH is adjusted to 7.0 with 1 N HCl prior to sterilization at 121° C.for 20 minutes. The cells are then plated out to select for mutants.

(b) Selection And Isolation

Mutagenized cells, as described above, are diluted and spread ontoplates containing a medium consisting of the following (modified fromFraser and Jerrel. 1963. J. Biol. Chem. 205: 291-295):

    ______________________________________                                        Glycerol             10.0   g/liter                                           K.sub.2 HPO.sub.4    0.5    g/liter                                           NH.sub.4 Cl          1.0    g/liter                                           MgSO.sub.4.7H.sub.2 O                                                                              0.5    g/liter                                           FeCl.sub.3.6H.sub.2 O                                                                              0.05   g/liter                                           Distilled Water, q.s.                                                                              1      liter                                             ______________________________________                                    

Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for30 minutes and then poured into sterile Petri plates.

Growth on this medium eliminates most nutritional auxotrophs produced bythe mutagensis procedure, e.g. cultures that require vitamins, growthfactors, etc. in order to grow on chemically defined medium areeliminated. After incubation at 28° C. for about 7 days, the resultingcolonies are replicated to test plates suitable for selecting mutantsand then back onto control plates containing the glycerol-based medium.The test plates are prepared as described by Peterson, G. E., H. L.Lewis and J. R. Davis. 1962. "Preparation of uniform dispersions ofcholesterol and other water-insoluble carbon sources in agar media." J.Lipid Research 3: 275-276. The minimal salts medium in these plates isas described above in section (a) of Example 1. Agar (15 g/liter), andan appropriate carbon source (1.0 g/liter), such as sitosterol orandrostenedione (AD), are added and the resulting suspension autoclavedfor 30 minutes at 121° C. The sterile, hot mixture is then poured into asterile blender vessel, blended for several minutes, and then pouredinto sterile Petri plates. Foaming tends to be a problem in thisprocedure but can be reduced by blending when the mixture is hot and byflaming the surface of the molten agar plates. In this manner uniformdispersions of water-insoluble carbon sources are obtained whichfacilitates the preparation of very homogenous but opaque agar plates.

Colonies which grew on the control plates, but which showed little ifany growth on test plates containing AD as the sole carbon source, arepurified by streaking onto nutrient agar plates. After growth at 28° C.,individual clones are picked from the nutrient agar plates with steriletoothpicks and retested by inoculating grided plates containing AD asthe carbon source. Purified isolates which still exhibit a phenotypedifferent from the parental culture are then evaluated in shake flasks.

(c) Shake Flask Evaluation

Shake flasks (500 ml) contain 100 ml of biotransformation mediumconsisting of the following ingredients:

    ______________________________________                                        Cerelose    5.0 g/liter                                                                              MgSO.sub.4.7H.sub.2 O                                                                     2.0 g/liter                                NH.sub.4 Cl 3.0 g/liter                                                                              UREA        0.5 g/liter                                KH.sub.2 PO.sub.4                                                                         0.5 g/liter                                                                              Tween 80*   2.0 g/liter                                CaCO.sub.3  3.0 g/liter                                                                              Ucon**      8.0 g/liter                                Na.sub.3 C.sub.6 H.sub.5 O.sub.7.2H.sub.2 O                                               3.0 g/liter                                                       ______________________________________                                         Tap H.sub.2 O to 1 liter, pH adjusted to 7.0 with 1N NaOH prior to            autoclaving.                                                                  *Atlas Refinery, Inc., Newark, New Jersey.                                    **Synthetic antifoam supplied by Union Carbide Chem. Co., NY, NY.        

Soyflour (1 g/liter) is blended into the medium and then sitosterol (30g/liter) is also blended into the medium. After the flasks areautoclaved for 30 minutes at 121° C., they are cooled to 28° C. and theninoculated with 10 ml of seed growth prepared as follows:

The purified isolates from part (b) are grown on agar slants at 28° C. Aloop of cells taken from a slant is used to inoculate a 500-ml flaskcontaining 100 ml of sterile seed medium consisting of the followingingredients:

    ______________________________________                                        Nutrient Broth                                                                           8 g/liter Glycerol       5 g/liter                                 (Difco)                                                                       Yeast Extract                                                                            1 g/liter Distilled Water, q.s.                                                                        1 liter                                   ______________________________________                                    

The pH is adjusted to 7.0 with 1 N NaOH prior to autoclaving the flasksat 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for72 hours.

As disclosed above, 10 ml of seed growth is then used to inoculate each500-ml flask containing 100 ml of sterile transformation medium. Theflasks are then incubated at 28° C. to 30° C. on a rotary shaker andsampled at various intervals. Ten ml samples are removed and extractedby shaking with 3 volumes of methylene chloride. Portions of theextracts are analyzed by thin layer chromatography using silica gel andthe solvent system described above, i.e., 2:3 (by volume) ethylacetae-cyclohexane, and by gas-liquid chromatography. Evidence of thepresence of ADD and small amounts of AD confirms the selectivedegradation of sitosterol by the mutant produced from the parent M.fortuitum ATCC 6842.

(d) Remutation To Give Mutant M. fortuitum NRRL B-11045

The above mutant which still grows very slowly on AD-containing agarplates, is then subjected to the mutation procedure, disclosed above, togive mutagenized cells. These cells are then subjected to the proceduresof parts (b) and (c), described above, to give the novel mutantMycobacterium fortuitum NRRL B-11045 which is capable of producing ADand small amounts of ADD.

EXAMPLE 2 Transformation of Sitosterol to AD

The medium used is the same as in Example 1 (c). This medium issterilized by autoclaving 30 minutes at 121° C., whereupon it is cooledto 30° C. and then inoculated with 10 parts of a seed culture of themutant mycobacterium M. fortuitum NRRL B-11045 prepared as described inExample 1 (c). The inoculated mixture is incubated at 30° C. for 336hours with agitation to promote submerged growth. Following incubation,the mixture is extracted with dichloromethane. The extract is dried overanhydrous sodium sulfate and the solvent is removed by vacuumdistillation. The resulting residue is dissolved in a minimum of ethylacetate-cyclohexane (20:80). This solution is then chromatographed onsilica gel. The presence of androst-4-ene-3,17-dione and a small amountof androsta-1,4-diene-3,17-dione (ratio of about 8:1) is shown by thinlayer chromatography. These compounds are separated from the silica gelby elution with the solvent system ethyl acetate-chloroform (15:85). Thecompounds are then isolated by evaporation of the solvent andrecrystallization from hexane.

EXAMPLE 3

By substituting cholesterol or sitosterol in Example 2 there is obtainedpredominantly AD and a small amount of ADD.

EXAMPLE 4

By substituting stigmasterol in Example 2 for sitosterol there isobtained predominantly AD and a small amount of ADD.

EXAMPLE 5

By substituting campesterol for sitosterol in Example 2 there isobtained predominantly AD and a small amount of ADD.

EXAMPLE 6

By adding a combination of any of the steroids in Examples 2-5, inaddition to sitosterol, or in place of sitosterol, in Example 2 there isobtained predominantly AD and a small amount of ADD.

EXAMPLE 7

By substituting a microorganism from the genera Arthrobacter, Bacillus,Brevibacterium, Corynebacterium, Microbacterium, Nocardia,Protaminobacter, Serratia, and Streptomyces, in Example 1 forMycobacterium fortuitum ATCC 6842, there are obtained mutantmicroorganisms which are characterized by their ability to selectivelydegrade steroids having 17-alkyl side chains of from 2 to 10 carbonatoms, inclusive, and accumulate predominantly AD in the fermentationbeer.

EXAMPLE 8

By substituting the mutants obtained in Example 7 for M. fortuitum NRRLB-11045 in Examples 2-6, there is obtained predominantly AD and a smallamount of ADD.

We claim:
 1. A process for preparing androst-4-ene-3,17-dione whichconsist of cultivating a mutant microorganism selected from the groupconsisting of Arthrobacter, Bacillus, Brevibacterium, Corynebacterium,Microbacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces,said mutant being characterized by its ability to selectively degradesteroids having 17-alkyl side chains of from 2 to 10 carbon atoms,inclusive, and accumulate androst-4-ene-3,17-dione in the fermentationbeer, in an aqueous nutrient medium under aerobic conditions in thepresence of a steroid containing from 2 to 10 carbon atoms, inclusive inthe 17-alkyl side chain and recovering androst-4-ene-3,17-dione.
 2. Aprocess, according to claim 1, wherein said mutant microorganism iscultivated in an aqueous nutrient medium under aerobic conditions in thepresence of a mixture of two or more steroids wherein each steroidcontains from 2 to 10 carbon atoms, inclusive, in the 17-alkyl sidechain.
 3. A process, according to claim 1, wherein said steroid isselected from the group consisting of sitosterol, cholesterol,stigmasterol, and campesterol.
 4. A process, according to claim 2,wherein said steroid mixture is selected from the group consisting ofsitosterol, cholesterol, stigmasterol, and campesterol.